Complete genome sequence of the dairy isolate Streptococcus macedonicus ACA-DC 198

Within the Streptococcus genus, only Streptococcus thermophilus is considered to be non-pathogenic due to its adaptation to the milk environment. Streptococcus macedonicus is also an intriguing streptococcal species since its most frequent source of isolation to date is fermented foods, mainly of dairy origin. Sequencing of S. macedonicus ACA-DC 198 genome was performed using a combination of 454 GS FLX pyrosequencing and HiSeq 2000 Illumina sequencing. The hybrid assembly between 454 and HiSeq2000 data (>200x coverage) resulted in one continuous genomic scaffold of 2,130,034 bp and a plasmid of 12,728 bp. The genome assembly was validated against a NheI optical map of the S. macedonicus genome. Sequences were annotated with the BaSys and the RAST pipelines and manually curated using Kodon. Final corrections were made based on the quality assessment of the annotation using GenePRIMP. We found 2,192 protein-coding genes on the chromosome, 192 of which were identified as potential pseudogenes, indicating an ongoing genome decay process. This hypothesis is also supported by the approximately 220 kb-smaller genome size of S. macedonicus compared to the S. gallolyticus genomes, despite the high level of gene synteny between the two species. Such a reductive evolutionary process is common for lactic acid bacteria domesticated to the food environment, which in the case of S. thermophilus was also accompanied by the loss of pathogenicity traits. With our in silico analysis we attempt to investigate whether S. macedonicus shows traits that would support its adaptation to the dairy environment at the genomic level

Ancient Origin and Gene Mosaicism of the Progenitor of Mycobacterium tuberculosis

The highly successful human pathogen Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success

First Worldwide Proficiency Study on Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Complex Strains

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.Institute for Public Health and the Environment (RIVM), European Centre for Diseases Prevention and Control (ECDC

Comparative analysis of pSMA198 found in Streptococcus macedonicus ACA-DC 198, the first streptococcal plasmid of the pCI305/pWV02 family of theta-replicating replicons

Here we analyze pSMA198, the first plasmid isolated from Streptococcus macedonicus ACA-DC 198, and we attempt to clarify the route of its original acquisition. Based on the similarity profiles of the plasmid’s replication initiation protein (Rep) and its origin of replication (ori), pSMA198 was found to be a novel member of the pCI305/pWV02 family of theta-replicating plasmids. The pCI305/pWV02 family consists of plasmids of narrow host range that are mainly found in lactococcal species. Comparative analysis of the pSMA198 revealed a high degree of similarity with plasmids pSK11b, pVF22 and pIL5 over its replication backbone, its mobilization backbone and most of its length, respectively. All these three plasmids have been isolated from Lactococcus lactis strains deriving from milk or its products supporting that S. macedonicus acquired pSMA198 from the latter species and that this acquisition took place in the dairy environment. Both pSMA198 and the chromosome of S. macedonicus exhibit a high degree of pseudogenes, indicating that they must have evolved under the same gene decay processes. Furthermore, we were able to determine chromosomal regions that may have originated from pSMA198, also supporting a long co-existence of the two replicons. In addition, pSMA198 is carried by S. macedonicus strains segregated in five different genotypes by pulsed-field gel electrophoresis (PFGE), showing that pSMA198’s acquisition is not a recent event. We propose that our overall analysis of pSMA198 points towards the habituation of S. macedonicus ACA-DC 198 to the dairy environment

Characterization of plasmid pSMA198 found in Streptococcus macedonicus ACA-DC 198 supports the relation of the species to the milk environment

Background: Streptococcus macedonicus is an intriguing streptococcal species whose most frequent source of isolation is fermented foods similarly to Streptococcus thermophilus. During the genome sequencing of S. macedonicus ACA-DC 198 a plasmid was identified. Objectives: To analyse pSMA198, the first plasmid isolated from S. macedonicus and to shed light onto its acquisition path. Methods: Similarity searches of nucleotide and protein sequences, comparative analysis of whole plasmid sequences and phylogenetic analysis were performed using the appropriate bioinformatics tools. Methods: Based on the similarity profiles of the plasmid’s replication initiation protein (Rep) and its origin of replication (ori), pSMA198 belongs to the narrow host range pCI305/pWV02 family found primarily in lactococci and it is the first such plasmid to be reported in streptococci. Comparative analysis of the pSMA198 over its ori, origin of transfer (oriT) or entire length revealed a high degree of similarity with plasmids pSK11b, pVF22 and pIL5, respectively, all isolated from Lactococcus lactis strains from milk or milk products. Phylogenetic analysis of the pSMA198 Rep showed that the vast majority of closely related proteins derive from lactococcal dairy isolates. Conclusions: Our findings demonstrate that S. macedonicus ACA-DC 198 acquired most probably plasmid pSMA198 from L. lactis during an ancestral genetic exchange event that took place in milk or dairy products. Based on our analysis we provide the first molecular and evolutionary evidence for the habituation of S. macedonicus to the dairy environment

Milk adaptation and pathogenic potential among members of the Streptococcus bovis/Streptococcus equinus complex

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) consists of species commonly found in the gastrointestinal tract (GIT) of herbivores. Certain members of the complex like Streptococcus macedonicus and Streptococcus infantarius are frequently isolated from traditional fermented foods, mostly of dairy origin. However, some species of this complex, like Streptococcus gallolyticus and Streptococcus pasteurianus are known pathogens of humans involved in a range of diseases including endocarditis, menengitis, bacteremia, colon cancer etc. Analysis of the genome of S. macedonicus strain ACA-DC 198 isolated from traditional Greek Kasseri cheese revealed important traits of adaptation to the dairy environment. The strain contains a significant percentage of potential pseudogenes indicating that it may have evolved through genome decay processes. Streptococcus macedonicus has an extra gene cluster for lactose and galactose metabolism unique among the SBSEC members and a typical proteolytic system required for casein hydrolysis. Furthermore, we found evidence in the genome of S. macedonicus suggesting HGT (horizontal gene transfer) events with potential donors Lactococcus lactis and Streptococcus thermophilus. Perhaps the most pronounced among these HGT events is the presence in S. macedonicus of pSMA198 which belongs to the lactococcal pCI305/pWV02 family of plasmids. Our analysis suggests that pSMA198 may have been acquired by S. macedonicus from L. lactis. Analogous traits have been reported for S. infantarius suggesting that at least these two strains present adaptations to the dairy environment among SBSEC. Finally, we were able to identify a number of potential virulence factors (VFs) within the SBSEC members whose presence varied among species of the complex. Streptococcus macedonicus and S. infantarius miss some of the VFs present in S. gallolyticus suggesting a diminished pathogenic potential for the two species

Comparative genomics of the dairy isolate Streptococcus macedonicus ACA-DC 198 against related members of the Streptococcus bovis/Streptococcus equinus complex

Background: Within the genus Streptococcus, only Streptococcus thermophilus is used as a starter culture in food fermentations. Streptococcus macedonicus though, which belongs to the Streptococcus bovis/Streptococcus equinus complex (SBSEC), is also frequently isolated from fermented foods mainly of dairy origin. Members of the SBSEC have been implicated in human endocarditis and colon cancer. Here we compare the genome sequence of the dairy isolate S. macedonicus ACA-DC 198 to the other SBSEC genomes in order to assess in silico its potential adaptation to milk and its pathogenicity status. Results: Despite the fact that the SBSEC species were found tightly related based on whole genome phylogeny of streptococci, two distinct patterns of evolution were identified among them. Streptococcus macedonicus, Streptococcus infantarius CJ18 and Streptococcus pasteurianus ATCC 43144 seem to have undergone reductive evolution resulting in significantly diminished genome sizes and increased percentages of potential pseudogenes when compared to Streptococcus gallolyticus subsp. gallolyticus. In addition, the three species seem to have lost genes for catabolizing complex plant carbohydrates and for detoxifying toxic substances previously linked to the ability of S. gallolyticus to survive in the rumen. Analysis of the S. macedonicus genome revealed features that could support adaptation to milk, including an extra gene cluster for lactose and galactose metabolism, a proteolytic system for casein hydrolysis, auxotrophy for several vitamins, an increased ability to resist bacteriophages and horizontal gene transfer events with the dairy Lactococcus lactis and S. thermophilus as potential donors. In addition, S. macedonicus lacks several pathogenicity-related genes found in S. gallolyticus. For example, S. macedonicus has retained only one (i.e. the pil3) of the three pilus gene clusters which may mediate the binding of S. gallolyticus to the extracellular matrix. Unexpectedly, similar findings were obtained not only for the dairy S. infantarius CJ18, but also for the blood isolate S. pasteurianus ATCC 43144. Conclusions: Our whole genome analyses suggest traits of adaptation of S. macedonicus to the nutrient-rich dairy environment. During this process the bacterium gained genes presumably important for this new ecological niche. Finally, S. macedonicus carries a reduced number of putative SBSEC virulence factors, which suggests a diminished pathogenic potential

Acquisition through Horizontal Gene Transfer of Plasmid pSMA198 by Streptococcus macedonicus ACA-DC 198 Points towards the Dairy Origin of the Species

Background: Streptococcus macedonicus is an intriguing streptococcal species whose most frequent source of isolation is fermented foods similarly to Streptococcus thermophilus. However, S. macedonicus is closely related to commensal opportunistic pathogens of the Streptococcus bovis/Streptococcus equinus complex. Methodology/Principal Findings: We analyzed the pSMA198 plasmid isolated from the dairy strain Streptococcus macedonicus ACA-DC 198 in order to provide novel clues about the main ecological niche of this bacterium. pSMA198 belongs to the narrow host range pCI305/pWV02 family found primarily in lactococci and to the best of our knowledge it is the first such plasmid to be reported in streptococci. Comparative analysis of the pSMA198 sequence revealed a high degree of similarity with plasmids isolated from Lactococcus lactis strains deriving from milk or its products. Phylogenetic analysis of the pSMA198 Rep showed that the vast majority of closely related proteins derive from lactococcal dairy isolates. Additionally, cloning of the pSMA198 ori in L. lactis revealed a 100% stability of replication over 100 generations. Both pSMA198 and the chromosome of S. macedonicus exhibit a high percentage of potential pseudogenes, indicating that they have co-evolved under the same gene decay processes. We identified chromosomal regions in S. macedonicus that may have originated from pSMA198, also supporting a long co-existence of the two replicons. pSMA198 was also found in divergent biotypes of S. macedonicus and in strains isolated from dispersed geographic locations (e.g. Greece and Switzerland) showing that pSMA198’s acquisition is not a recent event. Conclusions/Significance: Here we propose that S. macedonicus acquired plasmid pSMA198 from L. lactis via an ancestral genetic exchange event that took place most probably in milk or dairy products. We provide important evidence that point towards the dairy origin of this species

Predominance of Ancestral Lineages of Mycobacterium tuberculosis in India

Molecular epidemiologic findings suggest an ancient focus of TB

The Forest behind the Tree: Phylogenetic Exploration of a Dominant Mycobacterium tuberculosis Strain Lineage from a High Tuberculosis Burden Country

BACKGROUND: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. CONCLUSIONS/SIGNIFICANCE: Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications